This invention relates to a process for easily purifying human serum albumin obtained in high yields by gene manipulation. The present invention also relates to a composition comprising recombinant human serum albumin which shows an extremely low degree of coloring, which is a serious problem characteristic to recombinant human serum albumin.
Human serum albumin (hereinafter referred to simply as HSA) is the most abundant protein contained in plasma. It contributes to the maintenance of osmotic pressure in blood and binds to nutrients and metabolites to thereby transport these substances. HSA having these functions has been employed as a drug for treating hypoalbuminemia caused by an albumin loss or reduction in albumin synthesis, and in hemorrhagic shock.
HSA has been produced mainly from a fraction of collected blood. However, the process for producing HSA from blood has such problems as sporadic supply of blood, economical disadvantage, and contamination with undesirable substances such as hepatitis virus. Thus there has been an urgent requirement to develop a material usable as a substitute for naturally occurring HSA.
Under these circumstances, techniques for the mass production and purification of HSA by means of gene manipulation (as a substitute for the HSA originating in blood) have been developed as recombinant DNA technology has progressed.
To purify the HSA obtained by gene manipulation (hereinafter referred to as recombinant HSA and abbreviated as rHSA), it is not appropriate to apply the conventional processes for purifying HSA originating in plasma as such. This is because the impurities to be eliminated from rHSA completely differ from those contained in the HSA originating in plasma. Namely, rHSA is contaminated with, for example, coloring matters characteristic to recombinant HSA, proteins originating in the host cells, polysaccharides, etc. In particular, it is necessary to sufficiently eliminate components originating in the host cells, since they are foreign matters for living organisms including human being and thus can cause a problem of antigenicity.
Accordingly, there have been carried out various studies in order to isolate and purify to a sufficient degree rHSA produced via culture from components originating in the host cells and culture components. One of the conventional processes is exemplified by the process which comprises subjecting a yeast culture medium containing rHSA to pressingxe2x86x92ultrafiltration membrane treatmentxe2x86x92heatingxe2x86x92ultrafiltration membrane treatment and then treating by procedures such as chromatography using a cation exchanger and an anion exchanger, and hydrophobic chromatography [JP-A-5-317079 (the term xe2x80x9cJP-Axe2x80x9d as used herein means an xe2x80x9cunexamined Japanese patent applicationxe2x80x9d) corresponding to EP-A-570916, Biotechnology of Blood Proteins, 1993, 227, 293 -298]. Further, the process comprising the above-described procedure followed by the chelate resin treatment or the boric acid/borate treatment has been reported (EP-A-570916, JP-A-6-245789 corresponding to EP-A-612761).
In the above-mentioned conventional process, it is essentially required to effect the purification consisting of the above-mentioned several steps to thereby eliminate antigens originating in the host cells and achieve a high degree of purification. On the other hand, this process has such disadvantages as a decrease in the yield of rHSA and a prolonged treating period due to large number of steps. Although attempts have been made to elevate the yield of each step so as to improve the yield of rHSA, it seems that no further improvement in yield can be made and thus the yield of rHSA has already reached the upper limit. Moreover, the conventional process described above suffers from another problem that the pressing is effected in an open system and thus there is a risk of contamination. Namely, hygienic management, which is essentially required in the production of rHSA as a medicine, is highly difficult therein. In addition, the degree of coloring of rHSA can be reduced only to a A350/A280 ratio of about 0.015 (in the case of a solution containing 250 mg/ml of rHSA) at the lowest (JP-A-7-170993 and JP-A-7-170994 corresponding to EP-A-658569).
On the other hand, there has been developed a process for recovering a target protein directly from a crude culture medium without affecting any pre-treatment such as elimination of cells or concentration of the medium, after the completion of the cultivation (e.g., the streamline method with the use of expanded bed adsorption technique developed by Pharmacia, International Publication in Japan No. 6-500050 corresponding to EP-A-538467).
No case has been reported so far on the application of the above-mentioned expanded bed adsorption technique to the purification of rHSA, in particular, the recovery and purification of rHSA from a yeast culture medium. Thus, it remains unknown whether or not such a method is actually useful in the rationalization of the purification of rHSA and the improvement in the yield of the same. However, it is expected that the application of this method or one similar thereto to the purification of rHSA would contribute to the simplification of the conventional purification treatment consisting of several steps.
However, there arises a problem that, under the acidic conditions employed for the adsorption by a streamline column (adsorbent: Streamline SP) for use in the above-mentioned method, rHSA contained in the culture medium is rapidly degraded by proteases contained in the culture medium and thus the yield of rHSA is seriously lowered. It is, therefore, difficult to apply the above-mentioned expanded bed adsorption technique as such to the purification of rHSA.
Thus, there has been an urgent requirement to develop a process by which rHSA can be highly purified in a stable state at a high yield without spoiling the merits of the expanded bed adsorption technique (i.e., the simplification and rationalization of the purification process, etc.).
An object of the present invention is to provide a simple process for purifying recombinant HSA to a high purity level and to an excellent quality level within a short period of time. Another object of the present invention is to provide rHSA from which coloring matters characteristic to gene manipulation originating in the host, medium, etc. have been sufficiently eliminated, and a composition comprising the resulting rHSA.
To solve the above-mentioned problems, the present inventors have conducted extensive studies. As a result, they have found that proteases can be easily and effectively inactivated by directly heating a culture medium of an HSA production system in which host cells remain. Based on this finding, they have further found out that when this heated solution is contacted directly with adsorbent particles suspended in a fluidized bed without removing the cells therefrom, rHSA can be easily purified at a high yield. They have furthermore found out that the combination of the above-mentioned heating treatment with the adsorbent particle treatment makes it possible to reduce the number of steps of the conventional process for purifying rHSA from five (i.G., pressingxe2x80x94ultrafiltration membrane treatmentxe2x86x92heatingxe2x86x92ultrafiltration membrane treatmentxe2x86x92cation exchanger treatment) to two (heatingxe2x86x92adsorbent particle treatment), thus significantly shortening the purification period while elevating yield.
Moreover, the present inventors have found that their process makes it possible to obtain rHSA, which is substantially free from any impurities originating in the host cells and thus exhibits a substantially lowered degree of coloring compared with the one obtained by the conventional process.
Accordingly, the present invention relates to a process for purifying rHSA which comprises heating a culture medium containing rHSA and the rHSA-producing host, contacting the heated solution with adsorbent particles suspended in a fluidized bed and recovering the adsorbed fraction. More particularly, it relates to a process for purifying rHSA which comprises heating a culture medium containing rHSA and an rHSA-producing host, feeding the heated solution upward into a fluidized bed in which adsorbent particles are suspended to effect contacting the heated solution with the adsorbent particles, then reversing the flow direction and feeding downward a buffer to elute and recover the rHSA adsorbed by the adsorbent particles.
The present invention further relates to a purification process wherein a culture medium of an rHSA-producing host is heated to a temperature of from 50 to 100xc2x0 C. for from 1 minute to 10 hours, a process for purifying rHSA wherein a heated solution is contacted with adsorbent particles at a pH value of from about 3 to 5 in an atmosphere of an electric conductivity of from 0.1 to 50 mS, and a process for purifying rHSA wherein adsorbent particles are those having a strong cation exchange group.
The present invention further relates to a process for purifying rHSA wherein an adsorbed fraction containing rHSA, which has been recovered from the fluidized bed by the above-mentioned purification process, is subjected to at least one purification treatment selected from a group consisting of hydrophobic chromatography, anion exchanger treatment, chelate resin treatment, boric acid/borate treatment and ultrafiltration membrane treatment, preferably after heating in the presence of a reducing agent.
The present invention furthermore relates to a composition comprising recombinant human serum albumin which shows a A350/A280 ratio of below 0.015, when formulated into a 25% solution of the albumin (i.e., rHSA concentration: 250 mg/ml).